Recommended IP method for protein sample sizes <2 mL

While agarose/Sepharose® slurry and columns perform well for purifications of large amounts of protein or antibody, magnetic beads are best suited for the more-common, smaller-scale isolation of specific proteins and protein complexes. Protein research needs have changed since the introduction of Sepharose® in the 1970s, with the keys being a balance of capacity/yield, reproducibility, purity, and cost.

Over the past 25 years, streptavidin-coupled Dynabeads® in combination with a biotinylated ligand have been widely used and are cited in thousands of papers for diverse manual and automated applications. The most widely used application is for purification of nucleic acids (NA), but it is also used for immunoprecipitation (IP).

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Choose this if you've got a recombinant (fusion tagged) protein

Popular fusion tags for recombinant protein expression

His tag—consists of a string of six to nine histidine residues; primarily used for purification via immobilized metal affinity chromatography (IMAC)

His and GST are not true epitope tags, because they are not usually purified or detected via specific antibodies. By contrast, HA and c-Myc tags are true epitope tags, because their only means of purification or detection is via specific antibodies. Epitope tags are seldom used for bulk purification but are most often used for immunoprecipitation or co-immunoprecipitation (IP, co-IP). Nevertheless, all four of these tag systems can be used for either purification or pull-down applications.

If you’ve got a biotinylated antibody that recognizes the protein you wish to capture, you can create your own affinity product with one of these products.

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Dynabeads® magnetic beads comparison data

In comparison studies with Dynabeads® magnetic beads and immunoprecipitation kits from other manufacturers, kits offer excellent purification characteristics and experimental reproducibility in a shorter protocol time.

Immunoprecipitation was performed on Daudi cell lysate using 5 μg antibody with all products. 10% eluate from each IP experiment was loaded on a NuPAGE® 4–12% Bis-Tris and electrophoresed using MES running buffer. The resulting gel was stained using the SilverQuest ™ Silver Staining Kit (see below).

Protein elution properties

The recombinant protein A/G on the beads contains no albumin binding sites, thus avoiding co-purification of contaminating proteins. Binding of antibodies to the beads in solution is an equilibrium reaction. To capture as many antibodies as possible, the reaction volume should be kept low to maintain high concentrations of beads and antibody. There is no need for pre-treating the sample (even viscous samples), hence no dilution of sample or beads should be done. If the antibody concentration in the sample is suspected to be very low, the amount of beads can be increased.

As an alternative to eluting antibody from the beads, the Dynabeads® may be re-suspended in a Na-phosphate buffer. To prevent co-elution, it is possible to cross-link the antibody to protein A/G on the beads prior to immunoprecipitation. This is not necessary for downstream SDS-PAGE followed by autoradiography or western blotting, and optional for Silver or Coomassie® staining.

The publication trend for immunoprecipitation using magnetic separation is skyrocketing

*Note for mobile users: the interactivity of the Selection Guide for Immunoprecipitation does not function on mobile devices. To step through that video-based selection guide in your mobile device, use the links in the video description area (found below video play window).

High-capacity beads bind a broad range of antibodies from different species and subclasses

Optimized IP protocols and ready-to-go kits

Choices for non-covalent antibody attachment as well as covalent antibody and protein attachment

Mass spectrometry–compatible choices

Binds biotinylated proteins (can be used with recombinant antibodies that lack an Fc region)

Adaptable: one bead can be used for many assays, e.g., both nucleic acid and protein purification

Compatible with mass spectrometry

Good choice if sample contains free IgGs

Doesn’t require an antibody to isolate the target.

Same tag can be used on many different proteins

*Check the compatibility of your antibody with our kits using the Antibody Compatibility Table
†Crosslinking can be performed to avoid co-elution of the antibody, but this can decrease the yield of the target antigen.

† The stated binding strengths for Protein L refer only to antibody species and subtypes with appropriate kappa light chains. Lambda light chains and some kappa light chains do not bind.

For Igs with weak affinity to both protein A and protein G (or for Ig subclasses not specified in the table above), we recommend the Dynabeads® Antibody Coupling Kit, which is compatible with all antibody species and subclasses.

Note that products coated with pure Protein A or G are based on the Dynabeads® technology, while products coated with Protein A/G combination or Protein L are based on the Pierce magnetic bead technology.